The main goal of this proposal is to characterize by high-resolution, low-field 1H NMR, the proton bridges mobilized in the catalytic process in human thrombin, predominantly at the active site but also at subsites. It is hypothesized that short, strong hydrogen bonds (SSHBs) form at the active site when the catalytic His gets protonated in thrombin as in acid titration, or due to interactions with highly selective inhibitors. These are analogous to events at the transitions states (TS) in the course of catalysis of substrate hydrolysis. Most thrombin inhibitors in this study are targets of drug design for anticoagulant effects. SSHBs are believed to be an effective mode of acid-base catalysis employed by enzymes to reduce the TS free energy. Other specific sites critical for the allosteric function of thrombin are very likely to also have SSHBs. SSHBs will be characterized by internuclear hydrogen bond donor and acceptor distances < 2.6 [unreadable] in a linear H-bond, highly deshielded proton resonance signals, fractionation factors below unity and rapid exchange rates. The bond lengths will be calculated from both 1H NMR chemical shifts and fractionation factors. SSHBs will be studied each in the presence and absence of Na+ ions: in 1) pH titrations of thrombin in Tris buffer; 2) TS mimics in acylation, a) D-Phe-Pro-ArgCH2CI-inhibited, b) a peptidyl pyridinium methyl ketone-inhibited, with interactions at both active sites and subsites and c) a keto-amide transition state mimic complex with thrombin; 3) a mimic of the acylenzyme 4) three types of phosphonate ester covalent adducts of thrombin mimicking the TS for deacylation. 5) hirudin-inhibited thrombin with interactions at subsites and particularly at the anionic exosite. Binding of hirudin and its analogs to thrombin in the presence of viscogens and in heavy water will complement the NMR studies. A practical significance of these studies is the provision of guidelines for the design of mechanism-based or transition-state-analog inhibitors of thrombin. A great significance is also to basic understanding of enzyme catalytic power and enzyme-evolutionary theory.